Membrane-limited expression and regulation of Na+-H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct

1. Cell-specific reverse transcriptase-polymerase chain reaction (RT-POR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+-H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P-2 receptors in the duct. 2. The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM). 3. Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct. 4. Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1-like activity in the BLM and NHE2-like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present. 5. Activation of P-2 receptors resulted in activation of an NHE-independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+. 6. Stimulation of P-2U receptors expressed in the BLM activated largely NHE1-like activity, and stimulation of P-2Z receptors expressed in the LM activated largely NHE2-like activity. 7. The interrelation between basolateral and luminal NHE activities and their respective regulation by P-2U and P-2Z receptors can be used to co-ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.